Further advances in the genomics revolution, most importantly applications to medicine and diagnosis, will require low-cost, user-friendly, high-throughput and accurate tools for analyzing biomolecules. At the present time, the most pressing need is to reduce the consumption of biological samples and reagents. The bottleneck is not detection, since current equipment can work into the nano- to pico-molar range with nanoliter volumes, but rather fluid manipulation and the problem of introducing conveniently the samples and reagents in the system.
Robots, pipettors and microtiter plates (MTP) systems are generally limited to volumes above a few microliters by surface tension and evaporation.
A simple, robust method for handling sub-microtiter sample and reagent volumes and interfacing them with the outside world would thus have a tremendous impact on a wide range of high-throughput bioanalytical processes.
In the Bioanalyzer® device commercialized by Agilent, the sample is pipetted into vials in the microfluidic chip with conventional manual pipettors. Thus samples volumes of several μl are required.
Conventional capillary electrophoresis apparatuses, presently commercialized by Applied Biosystems, Beckman, Agilent and the like, collect samples by dipping a capillary end into either microvials such as “Eppendorf tubes” or microtiter plates.
Known methods however can lead to some carryover of liquid, because a drop of liquid, representing a volume much larger than the volume of liquid introduced into the capillary, can hang onto the capillary tip, and be transported to another vial.
To minimize this carryover, protocols generally involve a washing step, in which the capillary tip is dipped into a wash liquid contained in a second vial, before the tip is dipped into a buffer vial or another sample.
Patent US2002009392 to Jeffrey et al. discloses a method that involves a relative motion of the capillary tip and the wash liquid, which allows a better cleaning than a simple dipping. This method, however, involves a relatively long manipulation time. Also, this method does not solve the problem of evaporation, and thus requires relatively important sample volumes.
Litborn et al. in J Chromatogr B Biomed Sci Appl. 2000 Aug. 4; 745(1): 137-47 discloses to cover samples by a liquid lid consisting in a non-miscible, volatile fluid to prevent evaporation.
Another problem associated with the sipping of samples into capillaries is that when the tip is removed from a sample vial, because of hydrostatic forces, capillary forces and time constant of pressure regulation systems, either air can be spuriously injected into the channel, generating unwanted distorsion of flow and numerous possible inconveniences, or some fluid from the hanging drop may continue to be injected into the capillary, leading to irreproducible volume sampling.
To address this problem, U.S. Pat. No. 6,149,787 to Chow et al. discloses a method of sampling fluid comprising:                dipping an open end of an open ended capillary element comprising a capillary channel disposed therethrough, into a source of second fluid, the capillary channel being filled with a first fluid;        withdrawing the capillary element from the second fluid;        permitting an amount of the second fluid remaining on the open ended capillary to spontaneously inject into the capillary channel;        dipping the capillary element into a third fluid after a first selected time period, the first selected time period being controlled to control the amount of the first fluid permitted to spontaneously inject into the open ended capillary channel.        
This way, the quantity of sample introduced into the capillary is controlled thanks to the selected time period elapsed between dipping the capillary tip into the second fluid and into the third fluid. However, this method requires a precise control of the displacement of the tip, and it puts a lot of constraints on the injection process. For instance, if samples must be introduced from many different vials, such as in a microtiter plate, the injection time cannot be smaller than the time necessary for displacing the tip from the farthest vial to the buffer vial. Oppositely, if large samples must be injected, large waiting times must be used, which is detrimental to throughput.
Another difficulty in microcapillary and microfluidic systems is that transport of minute samples in thin capillaries leads to dispersion, cross contamination and dilution, due to the Poiseuille flow profile. A useful solution to this problem consists in transporting samples and reagents as droplets in an immiscible fluid.
Droplet systems, typically consisting of water droplets in oil or a fluorinated solvent, have received much attention in microfluidics as a method for producing precise emulsions, as discrete microreactors for polymerase chain reaction (PCR), for the measurement of fast kinetics, and for the dispersion-free transport and manipulation of sample aliquots.
Considerable efforts have thus been developed in the last years to create and/or manipulate microdroplets.
Some devices, such as disclosed in U.S. Pat. No. 6,130,098, for instance, use hydrophobic forces, by moving such droplets in microchannel combining some hydrophilic and some hydrophobic portions.
Manipulation of droplets on planar arrays of electrodes by electrowetting has also become very popular, since such manipulation allows one to address droplets to diverse locations and along complex and programmable paths.
Dielectrophoresis is another way of transporting and mixing droplets or solid objects such as cells or latex particles.
Transporting and mixing droplets in an elongated microchannel, or in a network of connected microchannels avoids evaporation and allows transport on long distances by simple hydrodynamic mobilization of a carrier fluid surrounding the droplets.
Droplets can thus be transported in capillaries several meters long, and used as microreactors, as disclosed e.g. in Curcio and Roeraade, Anal. Chem., 75, 1-7 (2003). When interaction with the walls is well controlled, all droplets move at the same velocity, and very stable trains are achieved, preventing unwanted mixing of neighboring droplets.
M. Curcio in Improved Techniques for High-Trough put Molecular Diagnostics, Ph. D. Thesis, discloses inter alia to serially inject reaction mixtures into a capillary tube as small plugs separated by a hydrophobic transport liquid to perform sequenced-flow PCR.
In some cases, however, it may be desirable to mix two droplets transported in a channel.
Patent application EP 04 2921734 to Viovy et al. provides a way to coalesce two droplets in a microchannel, using an electric field. This method, however, does not solve the world-to-chip interface, i.e. it does not provide a way to introduce the two reagents to be coalesced, in a way that would be both versatile, rapid, robust and convenient.